Natural sources of oils and fatty acids are derived from plants, animals and microorganism. The oils or lipids, thus produced from microorganisms are known as ?Single Cell Oil?. This present study aimed to isolate lipid accumulating bacteria for the production and optimization of single cell oil. In this preliminary investigation three soil samples were collected in around Kanchipuram District, 37 different bacterial strains were isolated and screened by Sudan black stain of that 3 strains (AOM13, AOM17 __ampersandsignamp; MEW3) showed maximum lipid accumulation. They were used for lipid production of by shake flask method. The lipid was extracted by Folch method using cell dry matters. Among 3 strains AOM17 was identified as the efficient producers which produce about 23% of lipid content. It was confirmed as lipid by solubility, saponification test. Further the lipid was subjected to antimicrobial activity against human pathogens by well diffusion method, the strain MEW3 showed good activity against Bacillus sp., Staphylococcus aureus and Proteus sp. In optimization, all three strains showed maximum lipid accumulation at 15.5 g/L of glucose, 0.4 g/L of ammonium sulphate, pH 8 and 96 hours incubation time. The three potential strains AOM13, AOM17 and MEW3 were identified as Cellulomanas sp., Arthrobacter sp., Acromicrobium sp., respectively. __ampersandsignnbsp;